PROJECT SUMMARY ? Overall Acute myeloid leukemia (AML) is one of the most common hematologic malignancies, representing a diverse collection of complex diseases. Treatment strategies for AML have not changed in 30-40 years. Single-agent targeted therapies based on AML genetics or microenvironmental disease features have been disappointing, at best. The investigators on this DRSC Program, Drug Combinations to Circumvent Resistance (D2CR), have collaborated for over a decade and have developed functional genomic pipelines for evaluation of primary AML patient samples that have collectively led to numerous discoveries with diagnostic and therapeutic implications. For this Program, our long-term goals are to translate effective drug combinations that target tumor- intrinsic and microenvironmental pathways into the clinic for patients with AML. Our immediate goals are to prioritize the most relevant tumor-intrinsic and microenvironmental pathways for each AML disease subset and establish sufficient preclinical data to facilitate immediate clinical investigation of drug combinations. Based on the central hypothesis that drug combinations targeting tumor intrinsic and extrinsic features of AML biology will be essential to the development of more effective and durable therapeutic strategies, we predict that the drug combinations established by this D2CR-DRSC Program will substantially improve outcomes for patients with AML. To accomplish these goals, 3 Projects are proposed: 1) What are the tumor-intrinsic genes and pathways that contribute to drug sensitivity and resistance? Genome-wide CRISPR/Cas screens on parental and drug-resistant AML cells will be integrated with computational analysis of the largest functional genomic AML cohort in the world. The result will nominate genes/pathways for validation on patient samples in gene-edited models and for drug combination studies in Project 3. 2) What are the tumor-extrinsic pathways promoting tumor cell growth, drug resistance, and immune suppression? Inflammatory cytokine profiling of our large bank of AML patient samples will be conducted. Our bank of AML patient bone marrow stromal cells will also be accessed for studies of the reactive signature of these stromal cells when exposed to specific drugs. Finally, high-parameter immunophenotyping and T-cell functional assays will be used to define the immune landscape with candidate drugs tested in an immune-competent, spontaneous mouse model of AML. Candidate targets will be nominated for combination studies with tumor-intrinsic targets. 3) What are the drug combinations that most effectively bridge tumor- intrinsic and microenvironmental biology to eliminate AML cells and circumvent resistance? Drug combinations from targets nominated in Project 1 and 2 will be tested ex vivo on primary AML patient samples and in vivo using AML patient-derived xenografts. Cumulatively, we expect these innovative analyses to have a major impact on our understanding of AML biology, with successful clinical translation of new, more effective drug combination strategies.